Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate

ABSTRACT

Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate are disclosed. More specifically, the present invention concerns inhibiting or preventing pyrophosphorolysis in sequencing and amplification of nucleic acid molecules. According to the present invention, an enzyme which is a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase, or a carboxy-lyase is added to the reaction which serves to remove pyrophosphate from the reaction mixture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional Application No.60/031,216, filed Nov. 22, 1996. The contents of this application arefully incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for nucleic acidsynthesis. Specifically, the present invention relates to DNA synthesisvia a primer extension reaction and methods for RNA synthesis. Inparticular, the invention relates to methods for avoiding the inhibitingeffects of pyrophosphate on RNA synthesis and primer extension DNAreactions, for example, polymerase chain reactions (PCRs) and sequencingreactions.

2. Background of the Invention

It has been recognized that pyrophosphorolysis, where an oligonucleotideis reduced in length, is detrimental to primer extension reactions. Thepyrophosphorolysis is caused by the availability of pyrophosphate. Forexample, PCR is inhibited by the addition of pyrophosphate even at verylow concentrations. According to U.S. Pat. No. 5,498,523, thispyrophosphorolysis can be prevented by providing an agent, for example,a pyrophosphatase, capable of removing pyrophosphate. Addition ofpyrophosphatase to a PCR greatly enhances the progress of the reactionand provides superior results compared to the reaction without apyrophosphatase. See U.S. Pat. No. 4,800,159 more uniformity inintensities of bands formed in a polyacrylamide gel used to identifyproducts of the sequencing reaction. This uniformity is due toprevention of degradation of specific DNA products bypyrophosphorolysis. See also, Tabor, S. and Richardson, C. C., J. Biol.Chem. 265:8322 (1990); U.S. Pat. No. 4,962,020; and Ruan, C. C. etal.,Comments 17(1):1 (1990).

Each product or band in a dideoxy sequencing experiment is apolynucleotide complementary to the template and terminated at the 3′end in a base-specific manner with a dideoxynucleotide. The dideoxystabilizes the product, preventing further polymerization of thepolynucleotide. However, in certain regions of the template, the bands,especially after prolonged reaction, will reduce in intensity orcompletely disappear (“drop-out” bands). A drop-out may not be readilydetected by the operator, leading to errors in the interpretation of thedata either by a human or computer-driven analyzer. Since thisphenomenon is stimulated by inorganic pyrophosphate, the effect ispresumably due to pyrophosphorolysis (reverse polymerization), not3′-exonucleolytic activity. It is hypothesized that DNA polymeraseidling at the end of these terminated products and in the presence ofsufficient pyrophosphate will remove the dideoxynucleotide, then extendfrom the now free 3′-hydroxyl end to another dideoxy termination. Ineffect, the bands are converted to longer polynucleotides/bands. Removalof pyrophosphate as it is generated in the polymerization reactioneliminates this problem.

Researchers have used a series of enzyme reactions coupled topyrophosphate generation to measure DNA polymers activity. In the first(P. Nyren, Anal. Biochem. 167:235 (1987)), Nyren used ATP: sulfateadenylyl-transferase to convert pyrophosphate and adenosine5′-phosphosulfate to ATP and sulfate ion. The ATP was used to make lightwith luciferase. In the second (J. C. Johnson et al., Anal. Biochem.26:137 (1968)), the researchers reacted the pyrophosphate withUDP-glucose in the presence of UTP: glucose-1-phosphateuridylyltransferase to produce UTP and glucose-1-phosphate. In two moresteps, polymerase activity was measured spectrophotometrically by theconversion of NADP to NADPH. While these articles describe the use ofATP: sulfate adenylyltransferase and UTP: glucose-1-phosphateuridylyltransferase in measuring DNA polymerase activity, they do notdescribe their use to prevent or inhibit pyrophosphorolysis in nucleicacid synthesis reactions.

SUMMARY OF THE INVENTION

A number of naturally-occurring enzymes use pyrophosphate as asubstrate, including certain transferases, kinases and lyases. Bycoupling the reaction catalyzed by one of these enzymes to thepolymerase reaction, pyrophosphate will not build up, preventingpyrophosphorolysis in nucleic acid synthesis reactions. Thus, thepresent invention relates to a method of inhibiting or preventingpyrophosphorolysis during synthesis of a nucleic acid molecule, saidmethod comprising

(a) combining one or more nucleotides and a nucleic acid template;

(b) incubating the one or more nucleotides and nucleic acid templatetogether with a polymerase and an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with alcoholgroup as acceptor, a nucleotidyltransferase, and a carboxy-lyase, underconditions sufficient to form a second nucleic acid moleculecomplementary to all or a portion of the nucleic acid template.

The method of the invention more specifically relates to a method ofinhibiting or preventing pyrophosphorolysis, said method comprising

(a) combining a primer with a nucleic acid template under conditionssufficient to form a hybridized product; and

(b) incubating said hybridized product in the presence of (i) one ormore nucleotides, (ii) a polymerase, and (iii) an enzyme selected fromthe group consisting of a pentosyltransferase, a phosphotransferase withalcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyaseunder conditions sufficient to synthesize a second nucleic acid moleculecomplementary to all or a portion of said nucleic acid template.

Specifically, the method of the present invention relates to inhibitionof pyrophosphorolysis in the synthesis of DNA and RNA molecules usingthe appropriate nucleotides and polymerases (dNTP's/rNTP's and DNApolymerase/RNA polymerase).

In particular, the present invention may be used in primer extensionreactions to prevent the inhibition of nucleic acid synthesis duringamplification and may be used to prevent band drop out in sequencingreactions. Thus, the invention relates to a method to prevent inhibitionof nucleic acid synthesis during amplification of a double strandednucleic acid molecule comprising

(a) providing a first and second primer, wherein said first primer iscomplementary to a sequence at or near the 3′ termini of the firststrand of said nucleic acid molecule and said second primer iscomplementary to a sequence at or near the 3′ termini of the secondstrand of said nucleic acid molecule;

(b) hybridizing said first primer to said first strand and said secondprimer to said second strand in the presence of (i) a polymerase, and(ii) an enzyme selected from the group consisting of apentosyltransferase, a phosphotransferase with an alcohol group as anacceptor, a nucleotidyltransferase and a carboxylyase under conditionssuch that a third nucleic acid molecule complementary to said firststrand and a fourth nucleic acid molecule complementary to said secondstrand are synthesized;

(c) denaturing said first and third strand and said second and fourthstrand; and

(d) repeating steps (a) to (c) one or more times.

The present invention also relates to a method of sequencing a DNAmolecule comprising:

(a) combining a primer with a first DNA molecule under conditionssufficient to form a hybridized product;

(b) contacting said hybridized product with nucleotides, a DNApolymerase, an enzyme selected from the group consisting of apentosyltransferase, a phosphotransferase with an alcohol group asacceptor, a nucleotidyltransferase and a carboxy-lyase; and a terminatornucleotide to give a reaction mixture;

(c) incubating the reaction mixture under conditions sufficient tosynthesize a random population of DNA molecules complementary to saidfirst DNA molecule, wherein said synthesized DNA molecules are shorterin length than said first DNA molecule and wherein said synthesized DNAmolecules comprise a terminator nucleotide at their 3′ termini; and

(d) separating said synthesized DNA molecules by size so that at least apart of the nucleotide sequence of said first DNA molecule can bedetermined.

The invention also relates to a solution for use in nucleic acidsynthesis, amplification or sequencing, comprising

(a) an enzyme selected from the group consisting of apentosyltransferase, a phosphotransferase with alcohol group asacceptor, a nucleotidyltransferase, and a carboxy-lyase;

(b) a substrate which is capable of either accepting a phosphate radicalfrom pyrophosphate or effecting transfer of pyrophosphate to give aphosphorylated product when in the presence of said enzyme; and

(c) a polymerase.

Examples of pentosyltransferases according to the present inventioninclude an adenine phosphoribosyltransferase or an orotatephosphoribosyltransferase. Examples of a phosphotransferase with alcoholgroup as acceptor include a pyrophosphate: glycerol phosphotransferase,a pyrophosphate: serine phosphotransferase, a pyrophosphate:fructose-6-phosphate 1-phosphotransferase or a pyrophosphate: purinenucleoside kinase. Examples of a nucleotidyltransferase include an ATP:sulfate adenylyltransferase, a UTP: glucose-1-phosphateuridylyltransferase or a glucose-1-phosphate adenylyltransferase.Examples of a carboxy-lyase include phosphoenolpyruvate carboxykinase.

The invention also relates to a kit comprising a container means havingin close confinement therein two or more container means, wherein afirst container means comprises an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with analcohol group as acceptor, a nucleotidyltransferase, and acarboxy-lyase; and optionally a nucleic acid polymerase. The polymerasemay instead be contained in a second container means. A third containermeans comprises a substrate which is capable of either accepting aphosphate radical to give a phosphorylated product from pyrophosphate oreffecting transfer of pyrophosphate radical when in the presence of theenzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a gel showing the effect of UTP: glucose-1-phosphateuridylyltransferase on pyrophosphorolysis in DNA sequencing reactions.

FIG. 2 depicts a gel showing the effect of ATP: sulfateadenylyltransferase on pyrophosphorolysis in DNA sequencing reactions.

FIG. 3 depicts a gel showing the effect of Pyrophosphate:fructose-6-phosphate 1-phosphotransferase on pyrophosphorolysis in DNAsequencing reactions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the recognition that there are anumber of enzyme reactions in a cell which utilize the high energy of aphosphodiester link in certain synthetic pathways, such as thosedescribed by Komberg, A and Baker, T. A., in DNA Replication, 2nd ed.,W. H. Freeman and Co., New York (1992), p. 68. These involve quitedifferent enzymes and reactions which are distinct from the degradativeinorganic pyrophosphorylase, but still prevent pyrophosphorolysis byreducing the level of pyrophosphate in the mixture. Some of the enzymetypes are transferases, kinases and lyases.

The following are official enzyme classes and particular examples ofenzymes that may be used in the practice of the invention: Class EC2.4.2.- Pentosyltransferases 2.4.2.7 Adenine phosphoribosyltransferase2.4.2.10 Orotate phosphoribosyltransferase Class EC 2.7.1.-Phosphotransferases with an alcohol group as acceptor 2.7.1.79Pyrophosphate: glycerol phosphotransferase 2.7.1.80 Pyrophosphate:serine phosphotransferase 2.7.1.90 Pyrophosphate: fructose-6-phosphate1-phosphotransferase 2.7.1.143 Pyrophosphate: purine nucleoside kinaseClass EC 2.7.7.- Nucleotidyltransferases 2.7.7.4 ATP: sulfateadenylyltransferase 2.7.7.9 UTP: glucose-1-phosphate uridylyltransferase2.7.7.27 ATP: glucose-1-phosphate adenylyltransferase Class EC 4.1.1.-Carboxy-lyases 4.1.1.38 Phosphoenolpyruvate carboxykinaseSee, the CRC Handbook of Biochemist and Molecular Biology: Proteins(Vol. II), Fasman, G. D., ed., 3rd edition, CRC Press, Cleveland, Ohio(1976), pp. 93-109, for the internationally developed classificationsystem for enzymes.

For comparison, inorganic pyrophosphatase belongs to a group verydistinct; hydrolases acting on phosphorous-maintaining acid anhydrides,with an EC classification number of 3.6.1.1.

There are many closely-related members of the 2.7.7.9 and 2.7.7.27enzymes types that may be used in the practice of the invention.Likewise, there are many members of the 2.4.2.—enzyme type that areclosely related enzymatically that may be used. Such additional enzymesare listed below:

Additional Members of the 2.4.2.—Subclass: E.C. 2.4.2.8 Hypoxanthinephosphoribosyltransferase E.C. 2.4.2.9 Uracil phosphoribosyltransferaseE.C. 2.4.2.11 Nicotinate phosphoribosyltransferase E.C. 2.4.2.12Nicotinamide phosphoribosyltransferase E.C. 2.4.2.14Amidophosphoribosyltransferase E.C. 2.4.2.17 ATPphosphoribosyltransferase E.C. 2.4.2.18 Anthranilatephosphoribosyltransferase E.C. 2.4.2.19 Nicotinate-nucleotidepyrophophorylase (carboxylating) E.C. 2.4.2.20 Dioxotetrahydropyrimidinephosphoribosyltransferase E.C. 2.4.2.22 Xanthine-guaninephosphoribosyltransferase

Additional Members of 2.7.7.—Subclass: E.C. 2.7.7.1Nicotinamide-nucleotide adenylyltransferase E.C. 2.7.7.2 FMNadenylyltransferase E.C. 2.7.7.10 UTP: hexose-1-phosphateuridylyltransferase E.C. 2.7.7.11 UTP: xylose-1-phosphateuridylyltransferase E.C. 2.7.7.13 Mannose-1-phosphateguanylyltransferase E.C. 2.7.7.14 Ethanolamine-phosphatecytidylyltransferase E.C. 2.7.7.15 Cholinephosphate cytidylyltransferaseE.C. 2.7.7.18 Nicotinate-nucleotide adenylyltransferase E.C. 2.7.7.21tRNA cytidylytransferase E.C. 2.7.7.23 Glucosamine-1-phosphateuridylyltransferase E.C. 2.7.7.24 Glucose-1-phosphatethymidylyltransferase E.C. 2.7.7.25 tRNA adenylyltransferase E.C.2.7.7.27 Glucose-1-phosphate adenylyltranferase E.C. 2.7.7.28Nucleoside-triphosphate-hexose-1-phosphate nucleotidyltransferase E.C.2.7.7.29 Hexose-1-phosphate guanylyltransferase E.C. 2.7.7.30Fucose-1-phosphate guanylyltransferase E.C. 2.7.7.32Galactose-1-phosphate thymidylyltransferase E.C. 2.7.7.33Glucose-1-phosphate cytidylyltransferase E.C. 2.7.7.34Glucose-1-phosphate guanylyltransferase E.C. 2.7.7.383-deoxy-manno-octulosonate cytidylyltransferase E.C. 2.7.7.39Glycerol-3-phosphate cytidylyltransferase E.C. 2.7.7.40D-ribitol-5-phosphate cytidylyltransferase E.C. 2.7.7.41 Phosphatidatecytidylyltransferase E.C. 2.7.7.42 Glutamate-ammonia-ligaseadenylyltransferase E.C. 2.7.7.43 Acylneuraminate cytidylyltransferaseE.C. 2.7.7.44 Glucuronate-1-phosphate uridylyltransferase E.C. 2.7.7.45Guanosine-triphosphate guanylyltransferase E.C. 2.7.7.46 Gentamycin2′-nucleotidyltransferase E.C. 2.7.7.47 Streptomycin3′-adenylyltransferase E.C. 2.7.7.50 mRNA guanylyltransferase E.C.2.7.7.52 RNA uridylyltransferase E.C. 2.7.7.54 Phenylalanineadenylyltransferase E.C. 2.7.7.55 Anthranilate adenylyltransferase E.C.2.7.7.57 N-methylphosphoethanolamine cytidylyltransferase E.C. 2.7.7.58(2,3-dihydroxybenzoyl)adenylate synthase E.C. 2.7.7.59 [Protein PII]uridylyltransferase

A number of such enzymes have been cloned and expressed in a recombinanthost. See, for example, Ladror, U. S. et al., J. Biol. Chem.266:16550-16555 (1991) (Pyrophosphate: fructose-6-phosphate1-phosphotransferase); Leyh, T. S. et al., J. Biol. Chem. 263:2409-2416(1988) (ATP: sulfate adenylyltransferase); Leyh, T. S. et al., J. Biol.Chem. 267:10405-10410 (1992) (ATP: sulfate adenylyltransferase);Weissborn, A. C., et al., J. Bacteriology 176:2611-2618 (1994)(UTP:glucose-1-phosphate uridylyltransferase); Allen, T. et al, Mol.Biochem. Parasitol. 74:99 (1995) (Adenine phosphoribosyltransferase);Vonstein, V. et al., J. Bacteriol. 177:4540 (1995) (Orotatephosphoribosyltransferase); Charng, Y. Y. et al., Plant Mol. Biol. 20:37(1992) (Glucose-1-phosphate adenylyltransferase); Kim, D. J. and Smith,S. M., Plant Mol. Biol. 26:423 (1994) (Phosphoenolpyruvatecarboxykinase); Jiang, Y. et al., Exp. Parasitol. 82:73 (1996)(Hypoxanthine-guanine phosphoribosyltransferase); Pla, J. et al., Gene165:115 (1995) (ATP phosphoribosyltransferase); Feldman, R. C. et al.,Infect. Immun. 60:166 (1992) (Uracil phosphoribosyltransferase);Vinitsky, A., J. Bacteriol. 173:536 (1991) (Nicotinatephosphoribosyltransferase); Ludin, K. M. et al., Curr. Genet. 25:465(1994) (Amidophosphoribosyltransferase); Rose, A. B. et al., PlantPhysiol. 100:582 (1992) (Anthranilate phosphoribosyltransferase);Hughes, K. T. et al., J. Bacteriol. 175:479 (1993) (Quinolatephosphoribosyltransferase); Jagadeeswaran, P. et al., Gene 31:309 (1984)(Xanthine-guanine phosphoribosyltransferase); Nakagawa, S., Biosci.Biotech. Biochem. 59:694 (1995) (FMN adenylyltransferase); Marolda, C.L. and Valvano, M. A., J. Bacteriol. 175:148 (1993) (Mannose-1-phosphateguanylyltransferase); Kalmar, G. B., Proc. Natl. Acad. Sci. USA 87:6029(1990) (Choline phosphate cytidylyltransferase); Muller-Rober, B. etal., Plant Mol. Biol. 27:191 (1995) (Glucose-1-phosphateadenylyltransferase); Shanmugam, K. et al., Plant Mol. Biol. 30:281(1996) (tRNA nucleotidyltransferase); Zapata, G. A. et al., J. Biol.Chem. 264:14769 (1989) (Acylneuraminate cytidylyltransferase); andVakylenko, S. B. et al., Antiobiot. Khimioter. 38:25 (1993) (Gentamycin2′-nucleotidyltransferase).

Preferred enzymes which may be used in the practice of the invention arethermostable, that is, they have been isolated from thermophilicorganisms or from recombinant host cells that have been transformed withDNA coding for the thermostable enzyme and derived from the thermophilicorganisms. Typically, the thermostable enzymes can withstandtemperatures above about 70° C. to about 100° C. for at least about aminute without losing substantially its enzymatic activity. Mostpreferably, the enzymes are obtained from extreme thermophiles and thethermostable enzyme is used in high temperature cycling reactions (e.g.PCR). Examples of such thermostable enzymes include phosphofructokinasefrom Thermoproteus tenax (Siebers, B. and Hensel, R. FEMS Microbiol.Lett. 111:1-8 (1993)); phosphofructokinase from Bacillusstearothermophilus (Zhu, X. et al., Biochem. 34:2560-5 (1995)(theorganism is not an extreme thermophile, the optimal performance withthis enzyme will be in the range 60-65° C.)); uridylyltransferase fromMethanococcus jannaschii (Bult, C. J. et al., Science 273:1058-1072(1996) (optimum temperature near 85° C.)); orotatephosphoribosyltransferase from Thermus thermophilus (Yamagishi, A etal., Appl. Environ. Microbiol. 62:2191-2194 (1996)); and uracilphosphoribosyltransferase from Bacillus caldolyticus (Ghim, S. Y. andNeuhard, J. et al., J Bacteriol. 176:3698-707 (1994)).

Of course, it is necessary to also employ a substrate which is capableof either accepting a phosphate radical to give a phosphorylated productfrom pyrophosphate or effecting transfer of pyrophosphate radical whenin the presence of the enzyme.

Examples of such enzyme/substrate combinations are shown in thefollowing Table. Enzyme Substrate Adenine phosphoribosyltransferase AMPOrotate phosphoribosyltransferase Orotidine 5′-phosphate Pyrophosphate:glycerol Glycerol phosphotransferase Pyrophosphate: fructose-6-phosphateD-Fructose-6-phosphate* 1-phosphotransferase Pyrophosphate: purinenucleoside kinase A purine nucleoside ATP: sulfate adenylyltransferaseAdenosine 5′-phosphosulfate UTP: glucose-1-phosphate Uridine5′-diphosphoglucose uridylyltransferase Glucose-1-phosphateadenylyltransferase ADP-glucose Phosphoenolpyruvate carboxykinaseOxaloacetate*Fructose-2,6-diphosphate may also be added, not as a substrate, but asa stimulator of enzyme activity.

Use of the enzyme/substrate combinations according to the presentinvention provide a method for preventing pyrophosphorolysis duringnucleic acid synthesis, amplification or sequencing. Thus, in oneembodiment, the invention relates to a method of inhibiting orpreventing pyrophosphorolysis during synthesis of a nucleic acidmolecule said method comprising:

(a) combining a primer with a first nucleic acid (DNA or RNA) templateto give a hybridized product; and

(b) incubating the hybridized product in the presence of (i) one or morenucleotides, (ii) a polymerase (DNA polymerase or RNA polymerase) and(iii) an enzyme selected from a group consisting of apentosyltransferase, a phosphotransferase with an alcohol group asacceptor, a nucleotidyltransferase, and a carboxy-lyase under conditionssufficient to synthesize a second nucleic acid molecule (RNA or DNA)complimentary to all or a portion of said nucleic acid template.

In a second embodiment, the present invention relates to a method ofsequencing a DNA molecule comprising:

(a) combining a primer with a first DNA molecule under conditionssufficient to give a hybridized product;

(b) contacting the hybridized product with one or more nucleotides, aDNA polymerase, an enzyme selected from the group consisting of apentosyltransfurase, a phosphotransferase with an alcohol group asacceptor, a nucleotidyltransferase, and a carboxy-lyase and a terminatornucleotide, to give a reaction mixture;

(c) incubating the reaction mixture under conditions sufficient tosynthesize a random population of DNA molecules complimentary to saidfirst DNA molecule, wherein said synthesized DNA molecules are shorterin length than said first DNA molecule and wherein said synthesized DNAmolecules comprise a terminator nucleotide at their 3′ termini; and

(d) separating said synthesized DNA molecules by size so that at least apart of the nucleotide sequence of said first DNA molecule can bedetermined.

In accordance with the present invention, it is possible to prevent banddrop-outs in DNA sequencing. Such band drop-outs occur to varyingextents in all known methods using any DNA polymerase. Thus, theinvention may be used to improve existing sequencing reactions forsingle-extension using e.g., modified T7 DNA polymerase, or for cyclesequencing, e.g., Taq-based cycle sequencing. Further, both radioactivelabeling and non-radioactive labeling methods are applicable. Forexample, Taq-based fluorescent sequencing in the Applied Biosystems DNASequencer, Model 373 or 377, will suffer errors resulting frompyrophosphorolysis without reducing the pyrophosphate generated in thereaction. Prolonged incubation seems more deleterious for sequencingbands, however, implementing this invention makes the sequencing processmore robust.

The dideoxy sequencing method was first described by Sanger et al.,Proc. Natl. Acad. Sci. USA 74:5463 (1977). Improvements andmodifications of the dideoxy sequencing method of Sanger et al. whichmay be used in the practice of the invention are described in U.S. Pat.No. 4,962,020, U.S. Pat. No. 5,498,523, U.S. Pat. Nos. 4,795,699,5,173,411, U.S. Pat. No. 5,405,746, U.S. Pat. No. 5,003,059, U.S. Pat.No. 5,409,811, U.S. Pat. No. 5,403,709, U.S. Pat. No. 5,405,747, U.S.Pat. No. 5,411,862, U.S. Pat. No. 5,432,065, U.S. Pat. No. 5,407,799,U.S. Pat. No. 5,525,464, U.S. Pat. No. 5,525,470, U.S. Pat. No.5,547,859, U.S. Pat. No. 5,503,980, U.S. Pat. No. 5,512,458, U.S. Pat.No. 5,308,751, U.S. Pat. No. 5,106,729, U.S. Pat. No. 5,124,247, U.S.Pat. No. 5,516,633, U.S. Pat. No. 5,484,701, U.S. Pat. No. 4,863,849,U.S. Pat. No. 5,332,666, U.S. Pat. No. 4,851,331, WO96/14434,WO95/20682, WO94/16107, WO95/23236, WO94/03643, WO93/04184, WO93/20232,WO93/05060, CA 1,311,201, and EP 0409 078. See also the M13Cloning/Dideoxy Sequencing Instruction Manual, BRL, Gaithersburg, Md.20884 (1980).

In accordance with the present invention, it is also possible to preventpyrophosphorolysis during amplification of nucleic acid molecules. Thus,the present invention relates to a method of preventing inhibition ofnucleic acid synthesis during amplification of double-stranded nucleicacid molecules comprising:

(a) providing a first and second primer, wherein said first primer iscomplementary to a sequence at or near the 3′ termini of the firststrand of said nucleic acid molecule and said second primer iscomplementary to a sequence at or near the 3′ termini of the secondstrand of said nucleic acid molecule;

(b) hybridizing said first primer to said first strand and said secondprimer to said second strand in the presence of (i) a polymerase, and(ii) an enzyme selected from the group consisting of apentosyltransferase, a phosphotransferase with an alcohol group as anacceptor, a nucleotidyltransferase and a carboxy-lyase, under conditionssuch that a third nucleic acid molecule complementary to said firststrand and a fourth nucleic acid molecule complementary to said secondstrand are synthesized;

(c) denaturing said first and third strand and said second and fourthstrand; and

(d) repeating steps (a) to (c) one or more times.

DNA polymerase enzymes that may be used according to the invention, e.g.dideoxy sequencing and PCR, include the wild type and mutant Tne DNApolymerases; Sequenase (T7 DNA polymerase), Taq DNA polymerase, ThermoSequenase, E. coli poll and Klenow fragment, AmpliTaq FS™, T5 DNApolymerase and mutants thereof Patents and patent applicationsdescribing these polymerases and others which may be used in thepractice of the invention include U.S. Pat. No. 5,270,179 U.S. Pat. No.5,466,591, U.S. Pat. No. 5,455,170, U.S. Pat. No. 5,374,553, U.S. Pat.No. 5,420,029, U.S. Pat. No. 5,075,216, U.S. Pat. No. 5,489,523, U.S.Pat. No. 5,474,920, U.S. Pat. No. 5,210,036, U.S. Pat. No. 5,436,326,U.S. Pat. No. 5,198,543, U.S. Pat. No. 5,108,892, U.S. Pat. No.5,192,674, U.S. Pat. No. 5,242,818, U.S. Pat. No. 5,413,926, U.S. Pat.No. 4,767,708, U.S. Pat. No. 5,436,149, U.S. Pat. No. 5,500,363, U.S.Pat. No. 5,352,778, U.S. Pat. No. 5,405,774, U.S. Pat. No. 5,545,552,WO96/14417, EP O 712 927, WO95/27067, WO91/09950, WO96/14405,WO95/14770, WO95/04162, WO92/06202, WO92/06188, EP O 482 714, EP O 701000, EP O 547, 359, EP O 386 859, EP O 386 858, WO96/10640, andapplication Ser. No. 08/706,702, filed Sep. 6, 1996, entitled “ClonedDNA Polymerases from Thennotoga maritima and Mutants Thereof ”Preferably, the DNA polymerase is a thermostable DNA polymerase such asTne, Taq, or Tma and mutants thereof which exhibit little or nodiscrimination between dideoxynucleoside triphosphates anddeoxynucleoside triphosphates, which exhibit little or no 3′ to 5′exonuclease activity, and which exhibit little or no 5′ to 3′exonuclease activity.

RNA polymerase enzymes that may be used according to the inventioninclude any one of the RNA polymerase I, II or III enzymes that aredescribed, for example, in U.S. Pat. Nos. 5,550,035, 5,102,802,5,122,457, 5,126,251, 4,952,496, 4,766,072, and 5,026,645; WO 95/15380,and WO 94/26911; and EP 647,716. Preferred RNA polymerase enzymesinclude SP6 RNA polymerase, T3 RNA polymerase and T7 RNA polymerasewhich are commercially available from Life Technologies, Inc.(Gaithersburg, Md.).

Chain terminators for DNA synthesis and sequencing reactions are listedin the following Table. Type of nucleoside triphosphate DNA polymerasetested Reference ribonucleoside 5′- Calf thymus TdT Beabealashvilli etal., Biochim. triphosphate Biophys. Acta 868: 136-144 (1986).ribonucleoside 5′- E. coli DNA Polymerase I Chidgeavadze et al.,Biochim. triphosphate Biophys. Acta 868: 145-152 (1986).arabinonucleoside 5′- Calf thymus TdT Beabealashvilli et al. Biochim.triphosphate Reverse transcriptase Biophys. Acta 868: 136-144 (1986).3′-amino-3′- T4 DNA Polymerase Chidgeavadze et al, Biochim.deoxyarabinonucleoside Biophys. Acta 868: 145-152 5′-triphosphate(1986). 2′-deoxy-2′- E. coli DNA Polymerase I Chidgeavadze et al.,Biochim. aminoribonucleoside Biophys. Acta 868: 145-152 5′-triphosphate(1986). 3′-azido-2′,3′- Reverse transcriptase Beabealashvilli et al.,Biochim. dideoxyribonucleoside Biophys. Acta 868: 136-1445′-triphosphate (1986). 3′-azido-2′,3′- E. coli DNA polymerase IPyrinova et al., Molekulyarnaya dideoxyribonucleoside Biologiya 22:1405-1410 (1988). 5′-triphosphate 3′-amino-2′,3′- E. coli DNA PolymeraseI Chidgeavadze et al., Nucl. Acids dideoxyribonucleoside Calf thymus DNAPol. α Res. 12: 1671-1686 (1984). 5′-triphosphate Rat liver polymerase β3′-amino-2′,3′- Calf thymus TdT Beabealashvilli et al., Biochim.dideoxyribonucleoside Reverse transcriptase Biophys. Acta 868: 136-1445′-triphosphate (1986). 3′-amino-2′,3′- E. coli DNA Polymerase IChidgeavadze et al., Biochim. dideoxyribonucleoside Calf thymus DNA Pol.α Biophys. Acta 868: 145-152 5′-triphosphate (1986).3′-N-acetylamino-2′,3′- Calf thymus TdT Beabealashvilli et al., Biochim.dideoxyribonucleoside 5′- Reverse transcriptase Biophys. Acta 868:136-144 triphosphate (1986). 3′-N-acetylamino-2′,3′- E. coli DNAPolymerase I Chidgeavadze et al, Biochim. dideoxyribonucleoside 5′-Biophys. Acta 868: 145-152 triphosphate (1986). 3′-fluorescaminyl-2′,3′-Reverse transcriptase Beabealashvilli et al., Biochim.dideoxyribonucleoside 5′- Biophys. Acta 868: 136-144 triphosphate(1986). 3′-fluorescaminyl-2′,3′- E. coli DNA Polymerase I Chidgeavadzeet al, Biochim. dideoxyribonucleaside 5′- Calf thymus DNA Pol. αBiophys. Acta 868: 145-152 triphosphate (1986).3′-N-biotinylamino-2′,3′- Calf thymus TdT Beabealashvilli et al.,Biochim. dideoxyribo-nucleoside Reverse transcriptase Biophys. Acta 868:136-144 5′-triphosphate (1986). 3′-N-biotinylamino-2′,3′- E. coli DNAPolymerase I Chidgeavadze et al, Biochim. dideoxyribo-nucleoside Calfthymus DNA Pol. α Biophys. Acta 868: 145-152 5′-triphosphate (1986).3′-amino-3′- Calf thymus TdT Beabealashvilli et al., Biochim.deoxyarabinonucleoside Reverse transcriptase Biophys. Acta 868: 136-1445′-triphosphate (1986). 3′-azido-3′- Reverse transcriptaseBeabealashvilli et al., Biochim. deoxyarabinonucleoside Biophys. Acta868: 136-144 5′-triphosphate (1986). 2′-deoxy-3′-O- Reversetranscriptase Beabealashvilli et al., Biochim. methylribonucleoside 5′-Biophys. Acta 868: 136-144 triphosphate (1986). 2′-deoxy-3′-O- AMVreverse transcriptase Kutateladze et al., FEBS Lett.methylribonucleoside 5′- 207: 205-212 (1986). triphosphate2′,3′-dideoxy-3′- E. coli DNA Polymerase I Chidgeavadze et al., FEBSLett. fluororibonucleoside 5′- AMV reverse transcriptase 183: 275-278(1985). triphosphate Calf thymus TdT 2′,3′-dideoxy-3′- Reversetranscriptase Beabealashvilli et al., Biochim. fluororibonucleoside 5′-Biophys. Acta 868: 136-144 triphosphate (1986). 2′,3′-dideoxy-3′- T4 DNAPolymerase Chidgeavadze et al, Biochim. fluororibonucleoside Biophys.Acta 868: 145-152 5′-triphosphate (1986). 2′,3′-didehydro-2′,3′- E. colipolymerase I KF Dyatkina et al., FEBS Lett. dideoxyribonucleoside 5′-Rat liver DNA polymerase β 219: 151-155 (1987). triphosphate AMV reversetranscriptase RSV reverse transcriptase Calf thymus TdT 3′-chloro-2′,3′-E. coli DNA polymerase I, Krayevsky et al., Nucleosidesdideoxyribonucleoside 5′- Rat liver DNA Polymerase β and Nucleotides 7:613-617 triphosphate AMV reverse transcriptase (1988). RSV reversetranscriptase Calf thymus TdT 3′-methylsulfonamido-2′, AMV reversetranscriptase Krayevsky et al., Nucleosides 3′-dideoxy- and Nucleotides7: 613-617 ribonucleoside 5′- (1988). triphosphate 2′,3′-di-O- AMVreverse transcriptase Krayevsky et al., Nucleosidesisopropylideneribonucleoside and Nucleotides 7: 613-617 5′-triphophate(1988).

The term “nucleotide” includes deoxyribonucleoside triphosphates such asdATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof Suchderivatives include, for example [αS]dATP, 7-deaza-dGTP and7-deaza-dATP. The term “nucleotide” as used herein also refers toribonucleoside triphosphates (rNTPs) and their derivatives. Illustratedexamples of ribonucleoside triphosphates include, but are not limitedto, ATP, CTP, GTP, ITP and UTP.

This invention may also be used in methods where improvement ofsynthesis of nucleic acids by a polymerase is desired and wherepyrophosphorolysis is deemed counter-productive. Uses include:polymerase chain reaction, especially ‘Long PCR,’ and cDNA synthesis.Examples of patents describing these methods include U.S. Pat. No.4,965,188, U.S. Pat. No. 5,079,352, U.S. Pat. No. 5,091,310, U.S. Pat.No. 5,142,033, U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, U.S.Pat. No. 4,800,159, U.S. Pat. No. 5,512,462 and U.S. Pat. No. 5,405,776.In the case of cDNA synthesis, a reverse transcriptase polymerase isincubated with the MRNA template, the deoxynucleoside triphosphates andthe enzyme which prevents the build up of pyrophosphate.

The invention also relates to a kit comprising a container means such asa box having in close confinement therein two or more container meanssuch as vials, ampules, tubes, jars and the like, each of which containthe materials necessary to carry out the invention. For example, a firstcontainer means may comprise an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with analcohol group as acceptor, a nucleotidyltransferase, and acarboxy-lyase. This first container means may also comprise a DNA or RNApolymerase. Alternatively, a second container means may comprise the DNAor RNA polymerase. A third container means will comprise a substratewhich is capable of either accepting a phosphate radical to give aphosphorylated product from pyrophosphate or effecting transfer ofpyrophosphate when in the presence of the enzyme. Other container meansmay contain other reagents that are necessary for carrying out dideoxysequencing or PCR as are well known.

Preferably, the contents of the container means are present at workingconcentrations (e.g. 1×). Other container means may contain otherreagents that are necessary for carrying out dideoxy sequencing oramplification (PCR). Methods for preparing such compositions at workingconcentrations are described in application Ser. No. 08/689,815, filedAug. 14, 1996, entitled “Stable Compositions for Nucleic Acid Sequencingand Amplification.” The enzyme and substrate used for reducingpyrophosphate concentration may be mixed directly with the polymerase attheir appropriate concentrations, which in turn may be further mixedwith reaction buffer and nucleotides. In general, the enzymes andsubstrate are present at concentrations sufficient to reduce the levelof pyrophosphate in nucleic acid synthesis, amplification or sequencingreactions. Preferably, the enzymes and substrate are present atconcentrations which reduce the level of pyrophosphate and, as a result,prevent pyrophosphorolysis (e.g., reduce the inhibition of amplificationreactions and/or reduce or eliminate band drop out in sequencingreactions). Particular concentrations of the enzyme will vary accordingto the activity of the enzyme and the temperature of the reaction.Additionally, in high temperature cycling reactions (PCR), it may benecessary to add enzyme after each cycle if the enzyme is inactivated bythe cycle temperature. By way of illustration, when the enzyme is UTP:glucose-1-phosphate uridylyltransferase, the polymerase is SequenaseVersion 2.0, and the temperature of the reaction is 37° C., theconcentration of UTP: glucose-1-phosphate uridylyltransferase may rangefrom about 0.01 U/pl to about 15 U/μl, preferably about 0.15 U/μl. Theconcentration of the uridine 5′-diphosphoglucose may range from about 10μM to about 0.5 M, preferably about 190 μM. If instead the enzyme isATP: sulfate adenylyltransferase, the concentration of ATP: sulfateadenylyltransferase may range from about 0.001 U/μl to about 2 U/μl,preferably about 0.002 U/μl, and the concentration of adenosine5′-phosphosulfate may range from about 0.25 μM to about 0.5 M,preferably about 5 μM. If the enzyme is Pyrophosphate:fructose-6-phosphate 1-phosphotransferase, the concentration ofPyrophosphate: fructose-6-phosphate 1-phosphotransferase may range fromabout 0.00004 U/μl to about 4 U/μl, and the concentration offructose-6-phosphate may range from about 10 μM to about 0.5 M,preferably about 190 μM. The concentration of the stimulatory cofactorfructose-2,6-diphosphate may range from about 0.5 nM to about 100 μMpreferably about 50 nM.

Thus, the solution of the present invention is an aqueous and/orbuffered liquid containing the components described above. Thesecomponents are present in the solution at concentrations sufficient toperform their desired function. If the reaction mixture is intended toamplify a target nucleic acid molecule, the reaction mixture willcontain the enzyme which reduces the level of pyrophosphate, thesubstrate which is capable of either accepting a phosphate radical togive a phosphorylated substrate from pyrophosphate or effecting transferof pyrophosphate when in the presence of the enzyme, a DNA polymerase,all four dNTPs, the one or two oligonucleotide primers having a singlestranded region (and optionally a double stranded region) which arecapable of annealing to the target nucleic acid molecule, being extendedand thereby amplified. The primer extension reaction may also comprise achain terminator as described herein, e.g. a dideoxynucleosidetriphosphate, which allows for sequencing of the target DNA molecule bythe well known Sanger dideoxy sequencing method.

In general, the enzymes described in this invention are ubiquitous innature so different versions of any one enzyme could be obtained fromdifferent organisms for different reaction situations. For example, asub-room temperature reaction may be preferred, where an enzyme from acryophile may be appropriate. Alternatively, thermostable enzymes may beutilized. Further, many examples of enzymes using pyrophosphate as aco-substrate are known, so many different versions of this invention areanticipated. For example, the best conditions to enhance Long PCR maynot be the same as for DNA sequencing, thus a different enzyme may beneeded for each application. The variety of enzymes and sources permitsflexibility in optimal design of application of the invention.

The invention is based on enzymatic removal of the pyrophosphateconcomitant with nucleic acid synthesis. Some enzymes may not becompatible with the reaction environment preferred for nucleic acidsynthesis, e.g., pH, monovalent cation concentration, or Tris buffer.Many different enzymes exist which are anticipated to provide the neededreduction of pyrophosphate concentration. Thus, an appropriate enzymecan be found that would be compatible without requiring compromise ofoptimal nucleic acid synthesis conditions. Two examples are alreadyprovided in the literature (Nyren, ibid and Johnson et al, ibid).

In the Examples which follow, either UTP: glucose-1-phosphateuridylyltransferase and uridine-5′-diphosphoglucose, or ATP: sulfateadenylyltransferase and adenosine 5′-phosphosulfate, or Pyrophosphate:fructose-6-phosphate 1-phosphotransferase and fructose-6-phosphate, wereused in a Sequenase™ reaction incubated for 30 minutes without observingdrop-out bands. Without both enzyme and substrate, many drop-out bandsare evident in a 30-minute incubation.

Having now generally described the invention, the same will be morereadily understood through reference to the following Examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1 Use of UTP: glucose-1-Phosphate Uridylyltransferaseto Eliminate Pyrophosphorolysis in DNA Sequencing

Template and primer sufficient for 7 sequencing reactions were annealedin a 39 μl reaction volume by incubating for two minutes at 65° C. in aheating block and then slowly cooling the reaction to less than 37° C.The composition of the reaction was: 74 nM M13mp19(+) strand DNA, 90 nMM13/pUC 23 base Forward Sequencing Primer, 72 mM Tris-HCl (pH 7.5), 45mM NaCl, 18 mM MgCl₂. Six separate reactions were then radiolabeled byincubating the following 15.5 μl reactions at room temperature (23-24°C.) for 2 minutes: 26.5 nM M13Mp19(+) strand DNA, 32 nM M13/pUC 23 baseForward Sequencing Primer, 129 mM Tris-HCl (pH 7.5), 32 mM NaCl, 13 mMMgCl₂, 6.5 mM DTT, 0.32 μM (0.32 μCi/μl) [α-³⁵S]dATP, 0.194 μM dCTP,0.194 μM 7-deaza-dGTP, 0.194 μM dTTP, 0.21 U/μl Sequenase Version 2.0 T7DNA Polymerase. Additional components were present in the indicatedreactions at the concentrations given in the following table:Concentration of Concentration of Concentration of UTP: glucose-1-Inorganic uridine-5′- phosphate Reaction Pyrophosphatasediphosphoglucose* uridylyltransferase Number (U/μl) (μM) (U/μl)** 1 0 00 2 0.00004 0 0 3 0 320 0.016 4 0 320 0.0032 5 0 320 0.00065 6 0 00.0032*uridine-5′-diphosphoglucose (Cat. No. U-4625) Sigma**UTP: glucose-1-phosphate uridylyltransferase (Cat. No. U-5877) Sigma

Each mixture was then divided into four tubes for completion ofbase-specific termination reactions. These sets of reactions wereincubated for 30 minutes at 37° C. and had the following compositions in6 μl reaction volumes: 15.4 nM M13mp19(+) strand DNA, 18.5 nM M13/pUC 23base Forward Sequencing Primer, 75 mM Tris-HCl (pH 7.5), 40 mM NaCl, 7.5mM MgCl₂, 3.8 mM DTT, 0.19 μM (0.19 μCi/μl) [α-³⁵S]dATP, 33.3 μM dATP,33.4 μM dCTP, 33.4 μM 7-deaza-dGTP, 33.4 μM dTTP, 0.12 U/μl SequenaseVersion 2.0, T7 DNA Polymerase, and 3.3 μM ddATP, ddCTP, ddGTP or ddTTP.Additional components were present in the indicated reaction sets at theconcentrations given in the following table: Concentration ofConcentration of Concentration of UTP: glucose-1- Inorganic uridine-phosphate Reaction Pyrophosphatase 5′-diphosphoglucoseuridylyltransferase Number (U/μl) (μM) (U/μl) 1 0 0 0 2 0.00002 0 0 3 0187 0.0093 4 0 187 0.0019 5 0 187 0.00038 6 0 0 0.0019

Reactions were stopped by adding 4 μl of 95% formamide, 20 mM EDTA,0.05% bromophenol blue and 0.05% xylene cyanol FF and denatured for 2minutes at 70° C. Three-microliter aliquots were separated on a 6% TBE-7M urea sequencing gel. The dried gel was exposed to Kodak BioMAX x-rayfilm at room temperature for approximately 18 hours.

Results from these six sets of reactions are shown in the photograph ofthe. gel (FIG. 1), where each set are separately displayed, left toright, in the order 1 through 6 as described in the above table. Underconditions where neither UTP: glucose-1-phosphate uridylyltransferasenor inorganic pyrophosphatase were present in the reactions (set 1),certain sequencing bands are either faintly or not visible as indicatedby an asterisk on the figure. When UTP: glucose-1-phosphateuridylyltransferase is added at two different enzyme concentrations(sets 3-4), the bands are fully visible. In the presence of a lowerconcentration of this enzyme (set 5), only partial recovery of the bandsis seen. Even though UTP: glucose-1-phosphate uridylyltransferase waspresent in set 6, the absence of the enzyme substrate uridine5′-diphosphoglucose prevented the enzyme from reacting withpyrophosphate and providing protection from band loss. The inhibition ofpyrophosphorolysis demonstrated in this example is dependent on bothenzyme and enzyme substrate and is not the result of a contaminatingpyrophosphatase-type activity. For reference, the action of inorganicpyrophosphatase in protecting from band loss is shown in set 2.

Example 2 Use of ATP: Sulfate Adenylyltransferase to EliminatePyrophosphorolysis in DNA Sequencing

Template and primer sufficient for 7 sequencing reactions were annealedin a 70 μl reaction volume by incubating for two minutes at 65° C. in aheating block and then slowly cooling the reaction to less than 37° C.The composition of the reaction was: 41 nM M13mp19(+) strand DNA, 50 nMM13/pUC 23 base Forward Sequencing Primer, 200 mM Tris-HCl (pH 7.5), 50mM NaCl, 20 mM MgCl₂. Five separate reactions were then radiolabeled byincubating the following 15.5 μl reactions at room temperature (23-24°C.) for 2 minutes: 26.5 nM M13mp19(+) strand DNA, 32 nM M13/pUC 23 baseForward Sequencing Primer, 129 mM Tris-HCl (pH 7.5), 32 mM NaCl, 13 mMMgCl₂, 6.5 mM DTT, 0.32 μM (0.32 μCi/μl) [α-³⁵S]dATP, 0.194 μM dCTP,0.194 μM 7-deaza-dGTP, 0.194 μM dTTP, 0.21 U/μl Sequenase Version 2.0 T7DNA Polymerase. Additional components were present in the indicatedreaction sets at the concentrations given in the following table:Concentration of Concentration of Concentration of Re- Inorganicadenosine ATP: sulfate action Pyrophosphatase 5′-phosphosulfate*adenylyltransferase** Number (U/μl) (μM) (U/μl) 1 0 0 0 2 0.00004 0 0 30 8.4 0.0016 4 0 8.4 0.0032 5 0 0 0.0032*adenosine 5′-phosphosulfate (Cat. No. A-5508) Sigma**ATP: sulfate adenylyltransferase (Cat. No. A-8957) Sigma

Each mixture was then divided into four tubes for completion ofbase-specific termination reactions. These sets of reactions wereincubated for 30 minutes at 37° C. and had the following compositions in6 μl reaction volumes: 15.4 nM M13mp19(+) strand DNA, 18.5 nM M13/pUC 23base Forward Sequencing Primer, 75 mM Tris-HCl (pH 7.5), 40 mMNaCl, 7.5mM MgCl₂, 3.8 mM DTT, 0.19 μM (0.19 μCi/μl) [α-³⁵S]dATP, 33.3 μM dATP,33.4 μM dCTP, 33.4μM 7-deaza-dGTP, 33.4 μM dTTP, 0.12 U/μl SequenaseVersion 2.0 T7 DNA Polymerase, and 3.3 μM ddATP, ddCTP, ddGTP or ddTTP.Additional components were present in the indicated reactions at theconcentrations given in the following table: Concentration ofConcentration of Concentration of Inorganic adenosine ATP: sulfateReaction Pyrophosphatase 5′-phosphosulfate adenylyltransferase Number(U/μl) (μM) (U/μl) 1 0 0 0 2 0.00002 0 0 3 0 4.9 0.0093 4 0 4.9 0.0019 50 0 0.0019

Reactions were stopped by adding 4 μl of 95% formamide, 20 mM EDTA,0.05% bromophenol blue and 0.05% xylene cyanol FF and denatured for 2minutes at 70° C. Three-microliter aliquots were separated on a 6%TBE-7M urea sequencing gel. The dried gel was exposed to Kodak BioMAXx-ray film at room temperature for approximately 18 hours.

Results from these five sets of reactions are shown in the photograph ofthe gel (FIG. 2), where each set are separately displayed, left toright, in the order 1 through 5 as described in the above table. Underconditions where neither ATP: sulfate adenylyltransferase nor inorganicpyrophosphatase were present in the reactions (set 1), certainsequencing bands are either faintly or not visible as indicated by anasterisk on the figure. When ATP: sulfate adenylyltransferase is added(set 3), the bands are fully visible. In the presence of a lowerconcentration of this enzyme (set 4), only partial recovery of the bandsis seen. Even though ATP: sulfate adenylyltransferase was present in set5, the absence of the enzyme substrate adenosine 5′-phosphosulfateprevented the enzyme from reacting with pyrophosphate and providingprotection from band loss. Since the preparation of ATP: sulfateadenylyltransferase used in this example was contaminated withdeoxyribonucleases, the presence of some non-specific DNA fragment sizes(smearing) in the gel causes an increase in the background, but does notprevent the demonstration of the effectiveness of ATP: sulfateadenylyltransferase from removing pyrophosphate from the sequencingreactions. The inhibition of pyrophosphorolysis demonstrated in thisexample is dependent on both enzyme and enzyme substrate and is not theresult of a contaminating pyrophosphatase-type activity. For reference,the action of inorganic pyrophosphatase in protecting from band loss isshown in set 2.

Example 3 Use of Pyrophosphate: Fructose-6-Phosphate1-Phosphotransferase to Eliminate Pyrophosphorolysis in DNA Sequencing

Template and primer sufficient for 7 sequencing reactions were annealedin a 39 μl reaction volume by incubating for two minutes at 65° C. in aheating block and then slowly cooling the reaction to less than 37° C.The composition of the reaction was: 74 nM M13mp19(+) strand DNA, 90 nMM13/pUC 23 base Forward Sequencing Primer, 360 mM Tris-HCl (pH 7.5), 90mM NaCl, 36 mM MgCl₂. Seven separate reactions were then radiolabeled byincubating the following 15.5 μl reactions at room temperature (23-24°C.) for 2 minutes: 26.5 nM M13mp19(+) strand DNA, 32 nM M13/pUC 23 baseForward Sequencing Primer, 129mM Tris-HCl (pH7.5), 32mM NaCl, 13 mMMgCl₂, 6.5 mM DTT, 0.32 μM (0.32 μCi/μl) [α-³⁵S]dATP, 0.194 μM dCTP,0.194 μM 7-deaza-dGTP, 0.194 μM dTTP, 0.21 U/μl Sequenase Version 2.0 T7DNA Polymerase. Additional components were present in the indicatedreactions at the concentrations given in the following table:Concentration of Concentration Pyrophosphate: Concentration ofConcentration fructose-6- of Inorganic Fructose-6- of Fructose-2,6-phosphate 1- Reaction Pyrophosphatase phosphate* diphosphate**phosphotransferase*** Number (U/μl) (mM) (μM) (U/μl) 1 0 0 0 0 2 0.000040 0 0 3 0 0.32 0.09 0.0032 4 0 0.32 0.09 0.00065 5 0 0.32 0.09 0.00032 60 0 0.09 0.0032 7 0 0 0.09 0.00065*Fructose-6-phosphate (Cat. No. F-3627) Sigma**Fructose-2,6-diphosphate (Cat. No. F-7006) Sigma***Pyrophosphate: fructose-6-phosphate 1-phosphotransferase, from MungBean (Cat. No. F-8757)

Each mixture was then divided into four tubes for completion ofbase-specific termination reactions. These sets of reactions wereincubated for 30 minutes at 37° C. and had the following compositions in6 μl reaction volumes: 15.4 nM M13mp19(+) strand DNA, 18.5 nM M13/pUC 23base Forward Sequencing Primer, 75 mM Tris-HCl (pH 7.5), 40 mM NaCl, 7.5mm MgC₂, 3.8 mM DTT, 0.19 μM (0.19 μCi/μl) [α-³⁵S]dATP, 33.3 μM dATP,33.4 μM dCTP, 33.4 μM 7-deaza-dGTP, 33.4 μM dTTP, 0.12 U/μl SequenaseVersion 2.0 T7 DNA Polymerase, and 3.3 μM ddATP, ddCTP, ddGTP or ddTTP.Additional components were present in the indicated reactions at theconcentrations given in the following table: Concentration ofConcentration Concentration Pyrophosphate: Concentration of of Fructose-fructose-6- of Inorganic Fructose-6- 2,6- phosphate ReactionPyrophosphatase phosphate diphosphate 1-phosphotransferase Number (U/μl)(mM) (μM) (U/μl) 1 0 0 0 0 2 0.00002 0 0 0 3 0 0.19 0.05 0.0019 4 0 0.190.05 0.00038 5 0 0.19 0.05 0.00019 6 0 0 0.05 0.0019 7 0 0 0.05 0.00038

Reactions were stopped by adding 4 μl of 95% formamide, 20 mM EDTA,0.05% bromophenol blue and 0.05% xylene cyanol FF and denatured for 2minutes at 70° C. Three-microliter aliquots were separated on a 6% TBE-7M urea sequencing gel. The dried gel was exposed to Kodak BioMAX x-rayfilm at room temperature for approximately 18 hours.

Results from these seven sets of reactions are shown in the photographof the gel (FIG. 3), where each set are separately displayed, left toright, in the order 1 through 7 as described in the above table. Underconditions where neither Pyrophosphate: fructose-6-phosphate1-phosphotransferase nor inorganic pyrophosphatase were present in thereactions (set 1), certain sequencing bands are either faintly or notvisible as indicated by an asterisk on the figure. When Pyrophosphate:fructose-6-phosphate 1-phosphotransferase is added (set 3), the bandsare fully visible. In the presence of lower concentrations of thisenzyme (sets 4-5), only partial recovery of the bands is seen. Eventhough Pyrophosphate: fructose-6-phosphate 1-phosphotransferase waspresent at two different concentrations in sets 6 and 7, the absence ofthe enzyme substrate fructose-6-phosphate prevented the enzyme fromreacting with pyrophosphate and providing protection from band loss. Theinhibition of pyrophosphorolysis demonstrated in the example isdependent on both enzyme and enzyme substrate and is not the result of acontaminating pyrophosphatase-type activity. For reference, the actionof inorganic pyrophosphatase in protecting from band loss is shown inset 2.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof can make various changes andmodifications of the invention to adapt it to various usages andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

1. A method of inhibiting or preventing pyrophosphorolysis duringsynthesis of a nucleic acid molecule, said method comprising (a)combining one or more nucleotides and a nucleic acid template; and (b)incubating the one or more nucleotides and nucleic acid templatetogether with a polymerase and an enzyme selected from the groupconsisting of a pentosyltransferase, a phosphotransferase with alcoholgroup as acceptor, a nucleotidyltransferase, and a carboxy-lyase, underconditions sufficient to form a second nucleic acid moleculecomplementary to all or a portion of the nucleic acid template.
 2. Amethod of inhibiting or preventing pyrophosphorolysis during synthesisof a nucleic acid molecule, said method comprising (a) combining aprimer with a nucleic acid template under conditions sufficient to forma hybridized product; and (b) incubating said hybridized product in thepresence of (i) one or more nucleotides, (ii) a polymerase, and (iii) anenzyme selected from the group consisting of a pentosyltransferase, aphosphotransferase with an alcohol group as acceptor, anucleotidyltransferase, and a carboxy-lyase under conditions sufficientto synthesize a second nucleic acid molecule complementary to all or aportion of said nucleic acid template.
 3. The method of claim 2, whereinsaid enzyme of (b)(iii) is a pentosyltransferase.
 4. The method of claim3, wherein said enzyme is an adenine phosphoribosyltransferase or anorotate phosphoribosyltransferase.
 5. The method of claim 2, whereinsaid enzyme of (b)(iii) is a phosphotransferase with an alcohol group asacceptor.
 6. The method of claim 5, wherein said enzyme is apyrophosphate: glycerol phosphotransferase, a pyrophosphate: serinephosphotransferase, a pyrophosphate: fructose-6-phosphate1-phosphotransferase or a pyrophosphate: purine nucleoside kinase. 7.The method of claim 2, wherein said enzyme of (b)(iii) is anucleotidyltransferase.
 8. The method of claim 7, wherein said enzyme isan ATP: sulfate adenylyltransferase, a UTP: glucose-1-phosphateuridylyltransferase or an ATP: glucose-1-phosphate adenylyltransferase.9. The method of claim 2, wherein said enzyme of (b)(iii) is acarboxy-lyase.
 10. The method of claim 9, wherein said enzyme is aphosphoenolpyruvate carboxykinase.
 11. The method of claim 2, whereinsaid enzyme of (b)(iii) is a thermostable enzyme.
 12. The method ofclaim 2, wherein said nucleotide is a deoxyribonucleoside triphosphateand said polymerase is a DNA polymerase.
 13. The method of claim 1,wherein said nucleotide is a ribonucleoside triphosphate and saidpolymerase is an RNA polymerase.
 14. A method to prevent inhibition ofnucleic acid synthesis during amplification of a double stranded nucleicacid molecule, comprising (a) providing a first and second primer,wherein said first primer is complementary to a sequence at or near the3′ termini of the first strand of said nucleic acid molecule and saidsecond primer is complementary to a sequence at or near the 3′ terminiof the second strand of said nucleic acid molecule; (b) hybridizing saidfirst primer to said first strand and said second primer to said secondstrand in the presence of (i) a polymerase, and (ii) an enzyme selectedfrom the group consisting of a pentosyltransferase, a phosphotransferasewith an alcohol group as an acceptor, a nucleotidyltransferase and acarboxy-lyase under conditions such that a third nucleic acid moleculecomplementary to said first strand and a fourth nucleic acid moleculecomplementary to said second strand are synthesized; (c) denaturing saidfirst and third strand and said second and fourth strand; and (d)repeating steps (a) to (c) one or more times.
 15. The method of claim14, wherein said enzyme of (b)(ii) is a pentosyltransferase.
 16. Themethod of claim 15, wherein said enzyme is an adeninephosphoribosyltransferase or an orotate phosphoribosyltransferase. 17.The method of claim 14, wherein said enzyme of (b)(ii) is aphosphotransferase with an alcohol group as acceptor.
 18. The method ofclaim 17, wherein said enzyme is a pyrophosphate: glycerolphosphotransferase, a pyrophosphate: serine phosphotransferase, apyrophosphate: fructose-6-phosphate 1-phosphotransferase or apyrophosphate: purine nucleoside kinase.
 19. The method of claim 14,wherein said enzyme of (b)(ii) is a nucleotidyltransferase.
 20. Themethod of claim 19, wherein said enzyme is an ATP: sulfateadenylyltransferase, a UTP: glucose-1-phosphate uridylyltransferase oran ATP: glucose-1-phosphate adenylyltransferase.
 21. The method of claim14, wherein said enzyme of (b)(ii) is a carboxy-lyase.
 22. The method ofclaim 21, wherein said enzyme is a phosphoenolpyruvate carboxykinase.23. The method of claim 14, wherein said enzyme of (b)(ii) is athermostable enzyme.
 24. The method of claim 14, wherein said polymeraseis a DNA polymerase.
 25. A method of sequencing a DNA moleculecomprising: (a) combining a primer with a first DNA molecule underconditions sufficient to form a hybridized product; (b) contacting saidhybridized product with (i) nucleotides; (ii) a DNA polymerase; (iii) anenzyme selected from the group consisting of a pentosyltransferase, aphosphotransferase with an alcohol group as acceptor, anucleotidyltransferase and a carboxy-lyase; and a terminator nucleotideto give a reaction mixture; (c) incubating the reaction mixture underconditions sufficient to synthesize a random population of DNA moleculescomplementary to said first DNA molecule, wherein said synthesized DNAmolecules are shorter in length than said first DNA molecule and whereinsaid synthesized DNA molecules comprise a terminator nucleotide at their3′ termini; and (d) separating said synthesized DNA molecules by size sothat at least a part of the nucleotide sequence of said first DNAmolecule can be determined.
 26. The method of claim 25, wherein saidenzyme of (b)(ii) is a pentosyltransferase.
 27. The method of claim 26,wherein said enzyme is an adenine phosphoribosyltransferase or anorotate phosphoribosyltransferase.
 28. The method of claim 25, whereinsaid enzyme of (b)(iii) is a phosphotransferase with an alcohol group asacceptor.
 29. The method of claim 28, wherein said enzyme is apyrophosphate: glycerol phosphotransferase, a pyrophosphate: serinephosphotransferase, a pyrophosphate: fructose-6-phosphate1-phosphotransferase or a pyrophosphate: purine nucleoside kinase. 30.The method of claim 25, wherein said enzyme of (b)(iii) is anucleotidyltransferase.
 31. The method of claim 30, wherein said enzymeis an ATP: sulfate adenylyltransferase, a UTP: glucose-1-phosphateuridylyltransferase or an ATP: glucose-1-phosphate adenylyltransferase.32. The method of claim 25, wherein said enzyme of (b)(iii) is acarboxy-lyase.
 33. The method of claim 32, wherein said enzyme is aphosphoenolpyruvate carboxykinase.
 34. The method of claim 25, whereinsaid enzyme of (b)(iii) is a thermostable enzyme.
 35. The method ofclaim 25, wherein said nucleotides are deoxyribonucleoside triphosphatesand said polymerase is a DNA polymerase.
 36. A solution for use innucleic acid synthesis, amplification or sequencing, comprising (a) anenzyme selected from the group consisting of a pentosyltransferase, aphosphotransferase with alcohol group as acceptor, anucleotidyltransferase, and a carboxy-lyase; (b) a substrate which iscapable of either accepting either a phosphate radical to give aphosphorylated product from pyrophosphate or effecting transfer ofpyrophosphate when in the presence of said enzyme; and (c) a polymerase.37. The solution of claim 36, wherein said enzyme of (a) is apentosyltransferase.
 38. The solution of claim 37, wherein said enzymeis an adenine phosphoribosyltransferase or an orotatephosphoribosyltransferase.
 39. The solution of claim 36, wherein saidenzyme of (a) is a phosphotransferase with an alcohol group as acceptor.40. The solution of claim 39, wherein said enzyme is a pyrophosphate:glycerol phosphotransferase, a pyrophosphate: serine phosphotransferase,a pyrophosphate: fructose-6-phosphate 1-phosphotransferase or apyrophosphate: purine nucleoside kinase.
 41. The solution of claim 36,wherein said enzyme of (a) is a nucleotidyltransferase.
 42. The solutionof claim 41, wherein said enzyme is an ATP: sulfate adenylyltransferase,a UTP: glucose-1-phosphate uridylyltransferase or an ATP:glucose-1-phosphate adenylyltransferase.
 43. The solution of claim 36,wherein said enzyme of (a) is a carboxy-lyase.
 44. The solution of claim43, wherein said enzyme is a phosphoenolpyruvate carboxykinase.
 45. Thesolution of claim 36, wherein said enzyme of (a) is a thermostableenzyme.
 46. The solution of claim 36, wherein said polymerase is a DNApolymerase.
 47. The solution of claim 36, wherein said polymerase is anRNA polymerase.
 48. A kit comprising a container means having in closeconfinement therein two or more container means, wherein a firstcontainer means comprises an enzyme selected from the group consistingof a pentosyltransferase, a phosphotransferase with an alcohol group asacceptor, a nucleotidyltransferase, and a carboxy-lyase; and a thirdcontainer means contains a substrate which is capable of eitheraccepting a phosphate radical to give a phosphorylated product frompyrophosphate or effecting transfer of pyrophosphate when in thepresence of said enzyme; wherein a nucleic acid polymerase is optionallypresent in said first container means or is optionally comprised in asecond container means.
 49. The kit of claim 48, wherein said nucleicacid polymerase is present in said first container means.
 50. The kit ofclaim 48, wherein said nucleic acid polymerase is present in said secondcontainer means.
 51. The kit of claim 48, wherein said enzyme is athermostable enzyme.